MRI of myocardial infarction using lipid-based contrast agents

Ischemic heart disease is the leading cause of death worldwide. Occlusion of a coronary artery results in cardiac ischemia downstream, which leads to irreversible myocardial cell death. If patients survive the ischemic event, infarct healing and global cardiac remodeling take place. A dynamic cascade of events is initiated, which is characterized by four distinct phases: cell death, inflammation, the formation of granulation tissue and finally fibrosis (chapter 1). The main goal of this thesis was to develop and in vivo apply paramagnetic lipid-based contrast agents for contrast-enhanced MRI of murine myocardial infarction. The visualization of specific processes in myocardial infarction could give insight in pathophysiological mechanisms, predict outcome and provide readout of therapy efficacy. Furthermore, the nanoparticulate contrast materials are very promising as theranostics agents. Previously, several (targeted) contrast agents for the visualization of cell death, inflammation, angiogenesis and fibrosis have been developed. However, these are still not routinely applied. Moreover, MRI measurement protocols and sequences specifically aimed at contrast-enhanced MRI of the mouse heart have been developed. The current status of contrast-enhanced MRI of murine myocardial infarction is extensively reviewed in chapters 2 and 3. In chapter 4 the distribution and accumulation kinetics of paramagnetic micelles and liposomes in a mouse model of cardiac ischemia reperfusion (IR) injury was studied. In vivo T1-weighted MRI and high-resolution ex vivo fluorescence microscopy revealed that both types of nanoparticles accumulated specifically in the infarcted myocardium in both acute (day 1) and chronic (week 1 and 2) IR injury. Micelles displayed faster accumulation kinetics compared to liposomes, which is most probably related to their smaller size. Furthermore, liposomes sometimes co-localized with vessels and inflammatory cells, whereas this was not observed for micelles. Due to the specific accumulation in infarcted myocardium, the presented lipid-based nanoparticles are a promising platform for drug delivery to infarcted myocardium. Although reperfusion therapy limits infarct size, it also promotes apoptosis, resulting in adverse secondary IR injury. Visualization of apoptotic cells could aid in the detection of IR injury and the indication of potentially salvageable tissue. For this purpose, the potential of annexin A5 (anxA5)-functionalized liposomes was explored (chapter 5). AnxA5 is a protein that specifically binds to phosphatidylserine expressed by apoptotic cells. AnxA5-liposmes were injected in mice with IR injury and T1-weighted and cine MRI were performed 24 h later. Both anxA5-liposomes and non-functionalized liposomes accumulated in the infarcted myocardium, leading to similar signal intensities in the remote and infarct regions and a comparable distribution of enhanced pixels. Careful comparison of cine MR measurements and T1-weighted MR images revealed that anxA5-liposomes accumulated to a higher degree in less severely infarcted myocardium, whereas non-functionalized liposomes preferentially accumulated in severely infarcted myocardium. Ex vivo high-resolution microscopy confirmed these in vivo results. Therefore, anxA5-liposomes might be useful for drug delivery to potentially salvageable myocardial tissue. Inflammatory cells are key regulators in myocardial infarct healing and in adverse left ventricular remodeling. Therefore, a non-invasive method for imaging of inflammatory cells could provide information on infarct status and outcome. To this end paramagnetic phosphatidylserine (PS)-containing liposomes were developed. Inflammatory cells recognize PS expressed by apoptotic cells and subsequently engulf the dying cells. The association of liposomes to murine macrophages was determined in vitro (chapter 6). Liposomes containing 6 mol% of PS showed a higher association with macrophages compared to control-liposomes without PS. Furthermore, this association was Ca2+- and Mg2+-dependent and PS-containing liposomes were predominantly internalized by macrophages, whereas control-liposomes only bound to the macrophage cell membrane. Due to the enhanced in vitro uptake by macrophages, PS-containing liposomes might be suitable for in vivo visualization of macrophage content. Therefore, these liposomes were applied in a mouse model of IR injury (chapter 7). When PS-liposomes circulated for 2.5 h, the signal change on T1-weighted MR images was lower compared to mice injected with liposomes without PS. This is explained by the shorter blood-circulation half-life of PS-containing liposomes. Nevertheless, high-resolution ex vivo fluorescence microscopy revealed some co-localization of PS-liposomes and macrophages, while this was not observed for liposomes without PS. After 24 h of circulation both types of liposomes accumulated in the infarcted myocardium resulting in similar signal changes. These findings were confirmed by in vivo T1 mapping as well. Due to the specific association of PS-containing liposomes and inflammatory cells 2.5 h after administration, these nanoparticles might be suitable for drug delivery to inflammatory cells in the infarcted myocardium. Finally, chapter 8 concludes with a general discussion on the preceding chapters and some future perspective in the field of contrast-enhanced MRI of myocardial infarction